Automated systems and methods for preparing biological specimens for examination

ABSTRACT

The systems and methods disclosed herein permit automated preparation of biological specimens for examination. The disclosed systems and methods provide fast, efficient, and highly uniform specimen processing using minimal quantities of fluids. The methods include at least a fixing phase for fixing a biological specimen to a substrate such as a microscope slide, a staining phase for staining the specimen, and a rinsing phase for rinsing the specimen. One or more of the fixing, staining, and rinsing phases include one or more agitation cycles for distributing reagents evenly and uniformly across the specimen. The systems can be implemented as a standalone device or as a component in a larger system for preparing and examining biological specimens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part, and claims priority under 35U.S.C. §120, to U.S. patent application Ser. No. 12/943,687, filed onNov. 10, 2010. This application also claims priority under 35 U.S.C.§119(e)(1) to U.S. Provisional Patent Application No. 61/510,180, filedon Jul. 21, 2011. The contents of each of the foregoing applications areincorporated by reference in their entirety.

BACKGROUND

For years, laboratory technologists have used dyes and stains such asthose used in Romanowsky staining for preparing biological specimens toimprove the contrast of a specimen during examination. Such examinationtypically utilizes a microscope, another device that captures images ofthe specimen, or, in other instances, unaided visual examination.Several different systems and methods for preparing a specimen forexamination are known. For example, U.S. Pat. Nos. 6,096,271; 7,318,913;and 5,419,279, and published U.S. Patent Application Nos. 2008/0102006and 2006/0073074 relate to machines and methods for staining a substrateduring specimen processing. These publications provide various detailson staining and preparing specimens for examination.

SUMMARY

The present disclosure relates to automated systems and methods forpreparing biological specimens for examination. The specimens caninclude, for example, a blood sample containing red blood cells, whiteblood cells, and platelets, applied to a substrate, e.g., a microscopeslide or a cover slip. Different embodiments can be used to prepareother biological specimens from biological samples including bonemarrow, urine, vaginal tissue, epithelial tissue, tumors, semen, saliva,and other body fluids. Additional aspects of the disclosure includesystems and methods for fixing, staining, rinsing, and agitating thespecimens. In general, the systems and methods disclosed herein providefor rapid, efficient, and highly uniform specimen processing usingminimal fluid quantities. The methods include one or more fixing,staining, and rinsing phases, including one or multiple agitation phasesduring or after one or more of the fixing, staining, and rinsing phases.The systems can be implemented as a standalone device or as a componentin a larger system for preparing and examining biological specimens.

In general, in a first aspect, the disclosure features an apparatus forpreparing a biological specimen on a substrate for examination, theapparatus including: (a) a substrate arm including a substrate gripper;(b) a first actuator connected to the substrate arm and configured tomove the substrate arm between an open position and a specimenprocessing position; (c) a second actuator arranged and configured toagitate a substrate gripped by the substrate gripper on the substratearm; (d) a platform having a top surface located opposite the substratewhen the substrate arm is in the specimen processing position; and (e)two or more offsets arranged on the top surface of the platform suchthat when the substrate contacts all of the offsets in the substrateprocessing position, the substrate and top surface of the platform aresubstantially parallel and form a separation of at least about 50microns.

Embodiments of the apparatus can include any one or more of thefollowing features individually or in combination.

The first and second actuator can be the same actuator configured toboth move the substrate arm and to agitate a substrate gripped by thesubstrate gripper on the substrate arm. A total surface area of the topsurface of the platform can be smaller than a total surface area of thesubstrate. There can be at least three or more offsets arranged at outeredges of the top surface of the platform, where tips of the offsetsdefine a plane.

A suction port can be located on the substrate gripper; the suction portcan be connected to a suction source for providing suction to thesuction port through a suction tube, to thereby hold the substrate tothe substrate gripper. The apparatus can include a first stain portlocated on the top surface of the platform, a first stain reservoir, anda first stain conduit connected to the first stain port for providing afluid pathway for stain to be pumped from the first stain reservoir tothe first stain port and into the separation. The apparatus can includea second stain port located on the top surface of the platform at alocation different from the first stain port location, a second stainreservoir, and a second stain conduit, where both the first and secondstain ports are arranged on the top surface at a spacing from a specimenarea on the substrate when the substrate is in the specimen processingposition, and where the second stain conduit is connected to the secondstain port to provide a fluid pathway for stain to be pumped from thesecond stain reservoir to the second stain port and into the separation.

The apparatus can include a first fixative port located on the topsurface of the platform, a fixative reservoir, and a fixative conduitconnected to the first fixative port for providing a fluid pathway forfixative to be pumped from the fixative reservoir to the first fixativeport and into the separation. The apparatus can include a first rinseport located on the top surface of the platform, a rinse solutionreservoir, and a rinse tube connected to the first rinse port forproviding a fluid pathway for rinse fluid to be pumped from the rinsesolution reservoir to the first rinse port and into the separation.

The apparatus can include a first vacuum port located on the top surfaceof the platform, a first waste container, and a first waste conduitconnected to the first vacuum port for providing a pathway of negativepressure to evacuate fluid from the separation or substrate and depositthe fluid into the first waste container. The apparatus can include asecond vacuum port located on the top surface of the platform and asecond waste conduit connected to the second vacuum port for providing apathway of negative pressure to evacuate fluid from the separation orsubstrate and deposit the fluid into the first waste container. Thefirst and second vacuum ports can be located on opposite ends of the topsurface of the platform.

The platform can include: a fixative port; a first stain port; a secondstain port; a rinse port; a first vacuum port; and a second vacuum port.The apparatus can include a block arranged to support the platform,where the block includes: a fixative port; a first stain port; a secondstain port; a rinse port; a first vacuum port; and a second vacuum port,where each port on the block is in a location corresponding to a portlocated in the platform.

The apparatus can include: a first stain reservoir; a second stainreservoir; a fixative reservoir; a rinse solution reservoir; a wastecontainer; a pump; a plurality of fluid conduits connected to the pumpand to the reservoirs and arranged for dispensing fluid from any one ormore of the reservoirs; and a vacuum source for evacuating fluid fromthe substrate into the waste container. The apparatus can include adryer positioned to direct a flow of air across the specimen when thesubstrate is located in the open position.

Embodiments of the apparatus can also include any of the other features,and any combinations of features, disclosed herein, as appropriate.

In a further aspect, the disclosure features methods of preparing abiological specimen on a substrate for examination that include: (a)positioning the substrate with respect to a surface so that thebiological specimen faces the surface, and so that the substrate and thesurface are substantially parallel and form a separation of at leastabout 100 microns; (b) sequentially dispensing (i) a first fixativesolution, (ii) a first stain solution, (iii) a second stain solution,and (iv) a first rinse solution into the separation between thesubstrate and the surface in an amount sufficient to contact thespecimen and the surface; and (c) after dispensing each one of solutions(i), (ii), (iii), and (iv) in step (b), and before dispensing the nextone of solutions (i), (ii), (iii), and (iv) in step (b), performing atleast a first agitation cycle, where the first agitation cycle includeschanging the distance between the substrate and surface while thedispensed solution contacts the specimen for the duration of the firstagitation cycle, and removing the dispensed solution from the separationand from contacting the specimen.

Embodiments of the methods can include any one or more of the followingfeatures.

Each sequential dispensing step can include dispensing one of thesolutions in step (b) at a flow rate of at least 70 microliters persecond for no more than three seconds. The first agitation cycle caninclude increasing the distance between the substrate and the surface byat least ten microns, and decreasing the distance between the substrateand the surface by at least five microns. Removing the dispensedsolution can include applying a pressure of at least one pound persquare inch less than an atmospheric pressure to the separation for atleast two seconds.

Embodiments of the methods can also include any of the other featuresdisclosed herein, and any combination of features, as appropriate.

In another aspect, the disclosure features methods of preparing abiological specimen on a substrate for examination, where the methodsinclude: (a) positioning the substrate with respect to a surface so thatthe biological specimen faces the surface, and so that the substrate andthe surface are substantially parallel and form a separation of at leastabout 50 microns; (b) dispensing a first stain into the separationbetween the substrate and the surface in an amount sufficient to contactthe specimen and the surface; (c) performing at least a first agitationphase, wherein the first agitation phase includes changing the distancebetween the substrate and surface while the first stain is contactingthe specimen for the duration of the first agitation phase; and (d)removing the first stain from the separation and the specimen.

Embodiments of the methods can include any one or more of the followingfeatures.

The dispensing step can include dispensing the stain at a flow rate ofat least 70 microliters per second for no more than three seconds. Theagitation phase can include increasing the distance between thesubstrate and the surface by at least ten microns, and decreasing thedistance between the substrate and the surface by at least five microns.Removing the stain can include applying a vacuum force of at least onepound per square inch to the first stain in the separation for at leasttwo seconds.

The methods can include: dispensing a second stain into the separationbetween the substrate and the surface in an amount sufficient to contactthe specimen and the surface; performing a second agitation phase, wherethe second agitation phase includes changing the distance between thesubstrate and surface while the second stain is contacting the specimenfor the duration of the second agitation phase; and removing the secondstain from the separation and the specimen.

Embodiments of the methods can also include any of the other featuresand/or steps disclosed herein, and any combinations thereof, asappropriate.

In a further aspect, the disclosure features methods of preparing abiological specimen on a substrate for examination, where the methodsinclude: (a) positioning the substrate with respect to a surface so thatthe specimen faces the surface, and so that the substrate is positionedto form a separation between the surface and at least a portion of thesubstrate of at least about 50 to 250 microns, e.g., 50, 60, 65, 70, 75,80, 85, 90, 95, 100, 125, 150, 175, 200, 225, or 250 microns; (b)performing a fixing phase that includes (i) dispensing a fixative intothe separation between the substrate and the surface in an amountsufficient to contact the specimen and the surface, (ii) performing atleast a first agitation phase, where the first agitation phase includeschanging the distance between the substrate and surface while thefixative is contacting the specimen for the duration of the firstagitation phase, and (iii) removing the fixative from the separation andthe specimen; (c) performing a first staining phase that includes (i)dispensing a first stain into the separation between the substrate andthe surface in an amount sufficient to contact the specimen and thesurface, (ii) performing at least a second agitation phase, where thesecond agitation phase includes changing the distance between thesubstrate and surface while the first stain is contacting the specimenfor the duration of the second agitation phase, and (iii) removing thefirst stain from the separation and the specimen; (d) performing asecond staining phase that includes (i) dispensing a second stain intothe separation between the substrate and the surface in an amountsufficient to contact the specimen and the surface, (ii) performing atleast a third agitation phase, where the third agitation phase includeschanging the distance between the substrate and surface while the secondstain is contacting the specimen for the duration of the third agitationphase, and (iii) removing the second stain from the separation and thespecimen; and (e) performing a first rinse phase that includes (i)dispensing a first rinse into the separation between the substrate andthe surface in an amount sufficient to contact the specimen and thesurface, (ii) performing at least a fourth agitation phase, where thefourth agitation phase includes changing the distance between thesubstrate and surface while the first rinse is contacting the specimenfor the duration of the fourth agitation phase, and (iii) removing thefirst rinse from the separation and the specimen.

Embodiments of the methods can include any one or more of the followingfeatures.

The methods can include performing a second rinse phase, where thesecond rinse phase includes: (i) dispensing a second rinse into theseparation between the substrate and the surface in an amount sufficientto contact the specimen and the surface; (ii) performing at least afifth agitation phase, where the fifth agitation phase includes changingthe distance between the substrate and surface while the second rinse iscontacting the specimen for the duration of the fifth agitation phase;and (iii) removing the second rinse from the separation and thespecimen. The methods can further include performing a drying cycle bydirecting a flow of air across the specimen.

The combined method steps can be performed, for example, in less than 70seconds (e.g., in less than 60 seconds). In some embodiments, themethods can consume less than 650 microliters of fixative, first stain,second stain, and first rinse fluids. In certain embodiments, themethods can consume less than 850 microliters of fixative, first stain,second stain, first rinse, and second rinse fluids.

Embodiments of the method can also include any of the other featuresand/or steps, and any combinations thereof, disclosed herein, asappropriate.

In another aspect, the disclosure features automated specimenexamination systems that include: an applicator station that applies asample specimen to a substrate; any one of the biological specimenpreparation apparatus disclosed herein; and an imaging station thatimages the biological specimen after preparation by the specimenpreparation apparatus.

Embodiments of the automated specimen examination system can include anyone or more of the features disclosed herein, as appropriate, includingany one or more of the features of the biological specimen preparationapparatus' disclosed herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of an embodiment of an apparatus forpreparing biological specimens for examination, with both samplegrippers 20A and 20B in an open position.

FIG. 2 is another perspective view of a portion of the apparatus of FIG.1 (with the substrate arms and sample grippers not shown).

FIG. 3A is a further perspective view of the apparatus of FIG. 1, withsample gripper 20A in an open position and sample gripper 20B in aclosed (specimen processing) position.

FIG. 3B is a perspective view of an indexing mechanism of the apparatusof FIG. 1.

FIG. 4 is a perspective view of the apparatus of FIG. 1 showingconnections between the apparatus and fluid reservoirs by means ofmultiple fluid conduits.

FIG. 5 is a perspective view of a specimen examination system thatincludes an automated substrate mover and an embodiment of a specimenpreparation apparatus as described herein.

FIG. 6A is an expanded perspective view of a portion of the apparatus ofFIG. 1 showing specimen gripper 20B, platform 60B, and block 80B indetail.

FIG. 6B is a perspective view of a ball joint mechanism of the apparatusof FIG. 1.

FIG. 6C is a cross-sectional view of the ball joint mechanism of FIG.6B.

FIG. 7A is a flow chart showing a series of steps for moving substratearms from an open position to closed (specimen processing) position.

FIG. 7B is a schematic diagram of an embodiment of a specimenpreparation apparatus as described herein.

FIG. 8A is a flow chart showing an alternate series of steps for movingsubstrate arms from an open position to a specimen processing position.

FIG. 8B is a schematic diagram of an apparatus for preparing biologicalspecimens for examination that includes two actuators.

FIG. 9 is a flow chart showing a series of steps for applying fixativeto a specimen.

FIG. 10 is a flow chart showing a series of steps for applying stain toa specimen.

FIG. 11A is a flow chart showing a series of steps for removing excessfluid from a substrate.

FIG. 11B is a flow chart showing an alternate series of steps forremoving excess fluid from a substrate.

FIG. 12 is a flow chart showing a series of steps for rinsing aspecimen.

FIG. 13 is a flow chart showing a series of steps for agitating aspecimen.

FIG. 14 is a flow chart showing a series of steps for drying a specimen.

FIG. 15 is a perspective view of a specimen preparation apparatus asused in a larger specimen examination system.

FIG. 16 is a flow chart showing a series of steps for processing aspecimen mounted on a substrate.

FIG. 17 is a graph showing volume of fluid consumed as a function oftime in the flow chart of FIG. 16.

FIGS. 18A and 18B are perspective views of the apparatus of FIG. 1 thatshow placement of a substrate onto a substrate arm by an automatedsubstrate mover.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

Disclosed herein are methods and systems for automated biologicalspecimen processing. The automated specimen processing methods andsystems described herein provide advantages over manual and otherautomated processing methods, including enhanced processing speed whileusing minimal reagent volumes and concurrently producing a highlyuniform sample preparation that significantly reduces the variabilityassociated with the application of stains, fixatives, and other reagentsas compared to specimens processed by hand or by other systems.

Conventional automated processing methods typically have relatively highprocessing throughput while at the same time consuming large volumes ofprocessing fluids, or have relatively low processing throughput whileconsuming reduced volumes of fluids. For many applications, however,both high throughput operation and low fluid consumption are desirable.By maintaining high throughput, specimens can be efficiently processedfor subsequent examination. By keeping fluid consumption low, the amountof processing waste is reduced along with the required volume ofprocessing reagents, keeping operating costs low. The systems andmethods disclosed herein permit rapid automated processing of specimens(e.g., more than 100 specimens per hour by a single machine) using lowvolumes of processing fluids (e.g., less than 1 mL of fluids perspecimen), while producing highly uniform and repeatable results.

Biological Specimen Preparation Systems and Methods

Before specimens are examined, they are prepared in a series of steps toenhance the visual appearance of certain features in the specimens. FIG.1 illustrates an embodiment of an apparatus or machine 1 for preparing abiological specimen for examination or imaging on a substrate 2 such asa microscope slide, cover slip, or other transparent surface. Machine 1can be incorporated into an overall system for preparing and analyzingspecimens comprising body fluids or other biological samples containingcells, such as system 2000 shown in FIG. 15 and described below. Machine1 can generally include, or form a portion of, a system that features afirst station that obtains a specimen, a second station that applies thespecimen to a substrate, third and fourth stations for fixing andstaining the specimen, respectively, a fifth station that dries thespecimen, a sixth station that images the specimen, and a seventhstation for analyzing the images and data obtained from the specimen.Certain embodiments of machine 1 are compatible with system 2000; someembodiments of machine 1 can be used in other specimen preparationsystems, and/or as stand-alone devices.

Machine 1 can include or connect to a control system 5 as shown in FIG.4, which provides another perspective view of machine 1. Control system5 can include one or more computers each containing a central processingunit capable of executing software instructions stored on computerreadable media such as a hard drive, optical drive, or memory.Additionally, control system 5 can include electrical circuitry forexecuting the software instructions. Control system 5 can include a userinterface for receiving user commands to control the operation ofmachine 1. Software stored on or provided to the computer can includeprograms that control the operation of components of machine 1 duringspecimen processing, such as fluid pumps and vacuums. For example, thesoftware can include instructions for directing the machine 1 to applyvarious fixatives, stains, and rinses to the specimen, and to performseveral agitation steps during specimen processing.

In addition, the software can include default settings, and the userinterface may contain customization features for providing the user withthe ability to change these defaults settings. For example, the userinterface can contain customization features for allowing a user tocustomize the speed, frequency, or order of fixing, staining, andrinsing phases, as well as agitation parameters (further describedbelow). Control system 5 can also communicate via a network protocol(such as Appletalk®, IPX, or TCP/IP). For example, the network protocolmay use cables (such as twisted pair cables) and/or a wirelessconnection such as WiFi. The control system may be connected to alaboratory information system using the network protocol. The laboratoryinformation system can contain a server and/or database for storinginformation relating to specimens processed on machine 1. For example,the database may contain a table that provides information about theperson or source of the specimen (e.g., name, date of birth (DOB),address, time specimen was taken, gender, etc.), information relating toprocessing of specimen (processed on date ##/##/####, specimen number #,etc.), a copy of any images acquired of the specimen, and copies of anyresults obtained by analyzing the images.

Referring to FIG. 1, machine 1 can include supports 110A and 110B tosecure the device to a location within a system or a laboratoryworkstation. Machine 1 also includes one or more substrate arms 10A and10B, each connected at their base to an actuator 30A and 30B. Theopposite ends of the substrate arms 10A and 10B include substrategrippers 20A and 20B for receiving and holding substrates duringspecimen processing. Each substrate gripper 20A and 20B receives andholds a substrate 2 while machine 1 completes all specimen processingsteps (described below). The substrate may be or include a microscopeslide, a cover slip, or other transparent material suitable for holdinga specimen during specimen processing and microscopic examination afterspecimen processing. The embodiment of FIG. 1 depicts a glass microscopeslide, substrate 2, which includes a biological specimen 3. Usingsuction ports, substrate grippers 20A, 20B can hold the substrate 2 tosubstrate arms 10A, 10B during specimen processing. A suction tube 23provides suction to the substrate grippers 20A and 20B through suctionports 21A and 21B, and 22A and 22B (note that ports 21A and 22A arepositioned behind the slide 2 in FIG. 1, and are shown in dashed lines).

The machine 1 embodiment shown in FIGS. 1-3 is a dual substrate machine,capable of holding and processing a substrate on each of substrate arms10A and 10B. Other embodiments provide for processing a single substrateor three or more substrates, sequentially or simultaneously. Further,while the embodiments depicted in FIGS. 1-6 use suction to attach thesubstrates 2 to the substrate arms 10A and 10B, alternative embodimentscan use various types of clamps, fingers, or magnets (if the substrateis magnetized) to attach a substrate 2 to a substrate arm 10A duringspecimen processing.

In the embodiments shown in FIGS. 5 and 18A-B, machine 1 receives asubstrate 2 carrying a specimen 3 from an automated substrate mover 120or manually from an individual. As an example, the substrate mover 120can be a device that transports a substrate between stations (e.g.,station 121 to station 122 to station 123, to station 124, and tostation 125). FIG. 5 shows a system having a first label reader station121, an applicator station 122, a staining station 123 that includesmachine 1, a camera or imaging station 124, and a second label readerstation 125. The first label reader station 121 is configured to readinformation from substrate 2 such as a bar code and/or “fingerprint”information that is used to identify the particular substrate 2 andspecimen 3 thereon. The second label reader station 125 functions in thesame manner, and the information it reads is used to verify that thespecimen 3 that is imaged at station 124 is the same as the substratethat was processed.

Substrate mover 120 can include a gripper 127 for holding the substrate2, and registration circuitry or software to enable the mover 120 todetermine whether the substrate 2 is mounted in the mover 120. In oneembodiment, substrate mover 120 can include a hydraulic cylinder formoving substrate 2 from a first station 121 to a second station 122.After specimen processing, the substrate mover 120 may remove theprocessed substrate from staining station 123 and transport thesubstrate 2 to another station for substrate examination, such as amicroscope or station 124. Alternatively, an individual may manuallyremove a substrate from machine 1 after specimen processing.

The substrate arms 10A and 10B can rotate about an axis to enable thesubstrate to move from an open position for loading, to a specimenprocessing position, and back to the open position for unloading afterspecimen processing. FIG. 7A shows a flow chart 500 that includes aseries of steps for moving substrate arms from an open position to aprocessing position. Flow chart 500 is further described below withreference to FIG. 7B, which shows a schematic diagram of machine 1.

Note that machine 1 in FIG. 1 is configured to accept and examine twosubstrates. In the following discussion and figures, reference may bemade to only one set of components in machine 1 (e.g., substrate gripper20A, actuator 30A, substrate arm 10A, etc.). However, it is to beunderstood that the same steps, features, and attributes that aredisclosed in connection with one set of components can also apply to theother set of components in machine 1 (e.g., substrate gripper 20B,actuator 30B, substrate arm 10B, etc.). Thus, while the discussionherein focuses only on one set of components for clarity and brevity, itis understood that machines for specimen examination such as machine 1can include two or more than two sets of components, each set havingsome or all of the features discussed herein.

Returning to FIGS. 7A and 7B, in a first step 502 of flow chart 500,substrate mover 120 places a substrate 2 in contact with a substrategripper 20A. In step 504, substrate 2 is positioned on the substrategripper in a “specimen up” or “open” position. Next, in step 506,actuator 30A rotates substrate arm 10A by approximately 180° (see FIG.7B) to position substrate 2 in a “specimen down” or “specimenprocessing” or “closed” position (step 508), directly above platform60A, so that substrate 2 is in a processing position in step 510.

Then, in step 512, machine 1 stains specimen 3 positioned on substrate 2by directing suitable fluids including stains, wash fluids, andfixatives to be pumped from reservoirs 210A, 211A, 212A, and 213A intocontact with specimen 3 through ports 42A, 43A, 44A, and 45A. Excessfluids are removed from specimen 3 by vacuum pumping through ports 40Aand 41A, and are collected in waste collectors 230 and 231.

In step 514, following staining of specimen 3, actuator 30A rotatessubstrate arm 10 by approximately 180° (reversing the rotation of step506) to return the substrate to the “specimen up” position. Finally, instep 516, substrate mover 120 removes the processed substrate fromsubstrate gripper 20A. Other open or “specimen up” positions can also beused, provided that an operator or automated substrate mover can loadand unload substrates from machine 1. For example, the specimen upposition can be rotated 100° or more (e.g., 120° or more, 130° or more,140° or more) from the specimen processing position. In someembodiments, the specimen up position can be rotated less than 100°(e.g., less than 90°, less than 80°, less than 70°) from the specimenprocessing position, provided that an operator or substrate mover canload and unload substrates from machine 1.

Actuators 30A and/or 30B may include an electric motor, pneumatics,magnetic systems, or other hardware (e.g., a worm gear) to move arm 10Aand/or 10B. When substrate arms 10A and 10B are in an open position asdepicted in FIG. 1, grippers 20A and 20B can each receive a substrate 2.Once loaded onto a substrate gripper 20A or 20B, actuators 30A and/or30B then rotate arms 10A and/or 10B, and thus substrate 2, from the open(“specimen up”) position to a processing position (“specimen down,” asshown for arm 10B in FIG. 3A) for application of fixative, stain, andrinse solutions, including agitation steps, and back to an open positionfor unloading after processing.

With reference to FIG. 3A, actuator 30B has rotated substrate arm 10Bfrom the open position depicted in FIG. 1 to a “closed” or processingposition. FIG. 3A shows that the substrate 2 on substrate arm 10B hasbeen flipped over and rotated approximately 180° from its loadingposition shown in FIG. 1 to a downward-facing position where specimen 3on substrate 2 is substantially parallel to the surface of platform 60B.As discussed in connection with FIG. 7A above, while substrate 2 ispositioned proximal to platform 60B in the specimen processing positionshown, machine 1 applies various fixatives, stains, and rinses tospecimen 3 on substrate 2 through several processing phases, which willbe described in greater detail below. To remove substrate 2 from theprocessing position, actuator 30B rotates substrate arm 10B back to theopen position shown in FIG. 1 (both arms) and FIG. 3A (where only arm10A is in the open position).

In certain embodiments, control system 5 can detect the position of thearms utilizing one or more sensors 105A and 105B to detect indicatorarms 101A and 101B (as shown in FIGS. 1 and 3). Sensors 105A and 105Bcan be proximity sensors, e.g., photoelectric sensors, utilizing, e.g.,infrared light or various other technologies (lasers, motion detectors,etc.) to detect the presence or absence of the arms. For example,proximity sensors 105A or 105B can have a detection field, and thesensors can determine whether or not a substrate arm (e.g., arm 10Aand/or 10B) or a substrate gripper (e.g., gripper 20A and/or 20B) iswithin the detection field. Control system 5 can receive informationfrom the sensors to determine the positions of substrate arms 10. Forexample, when substrate arm 10B (not shown in FIG. 3A) is rotated to aprocessing position, proximity sensor 105B on the proximal end ofindicator arm 101B senses target substrate gripper 20B, and notifiescontrol system 5 that substrate arm 10B is rotated to a specimenprocessing position. In this position, proximity sensor 105B on thedistal end of indicator arm 101B will not send a signal to controlsystem 5, because the sensor does not detect any target (e.g., asubstrate arm or substrate gripper).

When substrate arm 10B rotates to an open position (as shown in FIG. 1),proximity sensor 105B on the distal end of indicator arm 101B sensestarget substrate gripper 20B, and notifies control system 5 thatsubstrate arm 10B is rotated to an open position. Stated differently,when substrate arm 10B has rotated away from the sensor 105B, thesensors send a “not present” signal to the control system 5. When arm10B is rotated into the open position, arm 10B is closer to the sensor105B, and the sensor can send a “present” signal to the control system5. In alternate configurations, the sensor can be mounted on substrate10B and can detect the presence of the indicator arm 101B. In someembodiments, control system 5 can be used to calibrate the position ofactuators 30A and 30B to known open and specimen processing positions,and/or to actively monitor the movement and position of substrate arms10A and 10B based on control signals and/or feedback received fromactuators 30A and 30B.

The structure and axis of rotation for substrate arms 10A and 10B inFIG. 1 may be varied in other embodiments of the invention. FIG. 8Ashows a flow chart 600 that includes an alternate series of steps formoving substrate arms from an open position to a processing position.Flow chart 600 is further described below with reference to FIG. 8B,which shows a schematic diagram of machine 1.

In step 602 of flow chart 600, substrate mover 120 places substrate 2 onsubstrate gripper 20A in a “specimen up” orientation. Then, in step 604,a first actuator 30A rotates substrate 2 by approximately 180° in aplane perpendicular to the plane of FIG. 8B, so that substrate 2 remainsoriented in a “specimen up” position above platform 60A. In step 606, asecond actuator 35A receives substrate 2 oriented in the “specimen up”position. Then, in step 608, second actuator 35A (e.g., positionedbetween substrate arm 10A and substrate gripper 20A) rotates thesubstrate 2 into a “specimen down” orientation. Second actuator 35A canalso move substrate 2 downward toward platform 60A so that substrate 2contacts offsets 70A and 70B.

Next, with substrate 2 in the processing position in step 610, machine 1stains specimen 3 on substrate 2 by applying stains, fixatives, and washsolutions as discussed above in connection with step 512 of flow chart500. After staining is complete, second actuator 35A rotates substrate 2from a “specimen down” orientation to a “specimen up” orientation (step614), and then first actuator 30A rotates substrate 2 by approximately180° (e.g., in a plane perpendicular to the plane of FIG. 8B, reversingthe rotation applied in step 606) so that the substrate remains orientedin a “specimen up” position. Finally, in step 618, substrate mover 120removes the processed substrate from substrate gripper 20A.

In general, machine 1 may include one or more (e.g., two, three, four,five, or more than five) platforms 60A and 60B as shown in FIGS. 1-3 forspecimen processing. As shown in FIG. 2, platform 60A can includelateral sides for supporting a top side of the platform. A shield 100,shown in FIGS. 1 and 3, can be positioned between the platforms 60A and60B to prevent fluids from splattering between the platforms 60. In someembodiments, shield 100 can be formed from a transparent material thatblocks fluids from one of platforms 60A and 60B from contaminating theother platform. In certain embodiments, shield 100 can be formed from amaterial that is translucent or opaque. In FIGS. 1 and 3, shield 100 isdepicted as being formed from a transparent material to allow othercomponents positioned behind shield 100 to be shown in the same figure.Shield 100 could also have been shown as being formed from an opaquematerial, in which case portions of some components such as platform 60Aand block 80A would have been obscured.

FIG. 3B shows an indexing mechanism 50A that can be used to translatethe machine 1 to provide substrates 2 from each of the substrategrippers 20A, 20B to a position for specimen processing. The indexingmechanism 50A can be in many forms, such as electromechanical devices(e.g., a rack and pinion gear set powered by an electric motor), linearactuators (e.g., pneumatic actuators, hydraulic actuators, orelectromagnetic actuators). Although, in the illustrated embodiment, theindexing mechanism 50A translates the machine 1 linearly between twopositions, other translation paths are possible based on the number ofplatforms included on the machine 1, and their configuration and layout,such as circular or semi-circular (e.g., an indexing table that can movein an arcuate path). As shown, the indexing mechanism 50A can include agear rack 50B attached to a base 50C of the machine 1 and a pinion gear50D attached to an electric motor 50E that is fixed to the base 50C. Themachine 1 can be attached to the base 50C using one or more slidingdevices 50F so that the machine 1 can move smoothly when translated bythe indexing mechanism 50A. During use, the indexing mechanism 50A canmove the machine 1 so that the multiple substrate grippers 20A and/or20B of the machine 1 to receive a substrate 2 from a substrate mover 120(shown in FIG. 5) so that a sample disposed on the substrate 2 can beprepared by the machine 1, and also so that, once prepared, thesubstrate gripper 20A and/or 20B can provide the substrate 2 having aprepared sample can be provided to the substrate mover 120 for sampleprocessing.

For machines having two platforms 60A and 60B, as in the illustratedembodiment, substrates 2 are typically provided to, and from, thesubstrate mover 120 in an alternating manner. In some embodiments, afirst substrate 2 is provided from the substrate mover 120 to a firstsubstrate gripper 20A, to be processed at a first platform 60A, whilethe machine 1 is in a first position. While the first substrate 2 isprocessed at the first platform 60A, the indexing mechanism 50A cantranslate the machine 1 to a second position so that a second substrategripper 20B can receive a second substrate, to be processed at thesecond platform 60B, from the substrate mover 120. While the secondsubstrate is processed at the second platform 60B, the indexingmechanism 50A can translate the machine 1 back to the first position sothat the substrate mover 120 can remove the first substrate 2 from thefirst substrate gripper 20A. Once the substrate 2 is removed from thefirst gripping platform 20A, a next substrate can be provided to thefirst gripping platform 20A. This method for providing substrates toalternating gripping platforms can be implemented for more than two(e.g., three, four, five, or more than five) platforms therebyincreasing throughput of specimens prepared for further evaluation.

Platforms 60A and 60B are typically formed from one or more materialsthat are relatively chemically inert with respect to the fluids usedduring specimen processing and provide a suitable surface tension.Exemplary materials that can be used to form platforms 60A and 60Binclude engineering thermoplastics, such as polyoxymethylene (e.g.,Delrin® manufactured by DuPont), high molecular weight fluorocarbons,such as polytetrafluoroethylene (PTFE) (e.g., Teflon® manufactured byDuPont), and metals such as aluminum, steel, and titanium, provided theyare manufactured and/or treated to provide a suitable surface tensionthat acts to assist in evenly distributing and confining the processingfluids to the space between substrate 2 and the platforms, and allowingsuitable evacuation of the processing fluids as well. By selection ofsuitable materials, the platforms can also advantageously reduce orminimize the formation of bubbles or spaces within the fluids as theyare distributed, and at the same time maintain a sufficient surfacetension such that fluid leakage out of the separation between theplatforms and substrate 2 is reduced or eliminated.

In general, the surface area of platforms 60A and 60B can be selected asdesired for purposes of substrate handling and fluid delivery. Factorssuch as the surface area of platforms 60A and 60B can also influence theselected surface area of substrate 2. For example, in some embodiments,the surface area of platform 60A (e.g., the area of the surface ofplatform 60A that faces substrate 2) is slightly smaller than the areaof the surface of substrate 2 that faces platform 60A. By maintainingsuch a relationship between the areas of the facing surfaces of platform60A and substrate 2, fluid leakage from the region between the surfacescan be reduced or eliminated. Typically, for example, the area of thesurface of substrate 60A that faces substrate 2 is smaller than the areaof the surface of substrate 2 by 2% or more (e.g., 3% or more, 5% ormore, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more,30% or more).

Platforms 60A and 60B can be attached to blocks 80A and 80B,respectively. Block 80A includes lateral sides 81A-84A supporting a topside 85A as shown in FIG. 2. Blocks 80A and 80B can be made of the sameor similar materials to those used for the platforms, including metals,ceramics, and/or plastics. Thus, materials such as Delrin® can be usedto form blocks 80A and 80B, particularly in embodiments that implementRomanowsky staining of specimens. Other materials that can be used inembodiments include metals, and Teflon® brandpolytetrafluoroethylene-coated aluminum, steel, or titanium.

In some embodiments, platforms 60A and/or 60B can be raised as shown inFIGS. 1-3. Alternatively, in certain embodiments, platforms 60A and/or60B can be flush with the upper surface of blocks 80A and 80B,respectively. In either case, certain features of machine 1 as well assurface tension of fluids and surface energy of the platform or blockprevent excess fluids from flowing past the edges of platforms 60A/60Band/or blocks 80A/80B.

As shown in FIGS. 1 and 2, platform 60A can include offsets 70A-70D toprovide a separation between the surface of platform 60A and substrate2, and prevent substrate 2 from contacting platform 60A. Platform 60Bcan include a corresponding set of offsets 71A-71D. Offsets can includestandoffs, pins, pegs, rods, beads, walls, or other structures thatprovide separation between the surface of platform 60A and/or 60B andsubstrate 2. Offsets 70A-70D and 71A-71D ensure that the surfaces ofplatforms 60A and 60B and substrate 2 remain substantially parallel whensubstrate 2 contacts the offsets. The benefit of maintaining these twosurfaces in parallel is that the volume enclosed between these twosurfaces is thus defined and can be precisely controlled. If the twosurfaces are not substantially parallel, and the angle between themchanges, then the volume between them also changes and is not fixed andprecisely controlled. In addition, the fluids may not apply uniformly tothe specimen if such two surfaces are not substantially parallel.

As used herein, the phrase “substantially parallel” means that twosurfaces are exactly parallel or nearly parallel, so that imperfectionsin the surface flatness of substrate 2 are reduced or eliminated whensubstrate 2 contacts the offsets. For example, although great care istaken in the production of substrates, certain substrates may haveimperfections such as twist and/or non-coplanar corners. In the systemsand methods disclosed herein, the use of offsets assists in correctingthese imperfections by improving the surface flatness of substrate 2where needed, orienting substrate 2 in a substantially parallelrelationship to platforms 60A and 60B in the process. The phrase“substantially parallel” covers situations in which the two surfaces arenot perfectly flat, but the offsets are all the same size or height, sothat at least the contact points of a surface of the substrate with theoffsets are in the same plane.

FIG. 6A shows substrate 2 with specimen 3 (specimen not shown),substrate gripper 20B, blocks 80A, 80B, platforms 60A, 60B, offsets70A-70D and 71A-71D, and separation 92 between substrate 2 and platform60B. Separation 92 allows fluids to travel between the surface ofplatform 60B containing ports 40B-45B and substrate 2 containingspecimen 3. The separation distance required for optimal specimenfixing, staining, and rinsing will vary depending on the flow rate offluids dispensed from ports 40B-45B (and/or ports 40A-45A), portdiameter, the viscosity of the fluids applied during processing, and theamount of suction available for removing fluids from the substrate,separation, and platform.

In some embodiments, for example, offsets providing a separation 92 ofabout 100-200 microns between the surface of platform 60B and substrate2 enable fixing, staining, and rinsing for specimens comprising bloodcells in embodiments capable of dispensing fluids at flow rates rangingfrom 70 to 140 microliters per second (e.g., 90, 115, or 125 microlitersper second) from ports 40B-45B having a diameter ranging from 500 to1,500 microns. In general, the size or height of separation 92 can varyfrom about 50 microns to 1,000 microns for certain embodiments (e.g.,from about 50 to 500 microns, from about 75 to 250 microns, from about100 to 200 microns), provided such embodiments are capable of overcomingsurface tension from fluids in the separation while dispensing andremoving fluid during specimen processing. In addition, in certainembodiments, the diameters of ports located on platform 60A and/or 60Bcan vary from about 125 microns to 5,000 microns.

FIGS. 6B and 6C show a ball joint mechanism 25 that can be used to aligna substrate gripper 20A to be parallel with a platform 60A. The balljoint mechanism 25 can include a ball member 25A that is rigidly fixedto the substrate gripper 20A, a deflection element 25B (e.g., a spring),a lower socket 25C that is rigidly connected to the substrate arm 10A,an upper socket 25D, a cap 25E that is fixed to the lower socket 25C(e.g., using fasteners), and a set screw 25F. In some embodiments,during manufacturing and/or set up of the machine 1 and substrategrippers 20A and/or 20B, the ball joint mechanism 25 can be adjusted tocompensate for any misalignment that may be present due to tolerancestack-up or fabrication problems. To adjust the ball joint mechanism 25,in some embodiments, the set screw 25F is loosened and the substrate arm10A is moved to the closed position. Since the set screw 25F isloosened, the substrate gripper 20A, while gripping a substrate 2, isable to lay substantially parallel to the platform 60A while thesubstrate 2 positioned along the contact offsets 70. Alternatively, insome embodiments, the number of offsets on platform 60 can be reduced oreliminated completely; a shim with a thickness corresponding to thedesired separation distance can be used temporarily during set up orcalibration of machine 1 in conjunction with ball joint mechanism 25 toset separation 92 at a desired distance for specimen processing.Although the ball joint mechanism 25 is loosened, the deflection element25B applies a force to keep the substrate gripper 20A semi-fixed to thesubstrate arm 10A so that it is able to move independently, but it isnot so loose and not free to move so much as to interfere with, or causedamage to, other components of the machine 1. Once the substrate 2 ispressed firm in a closed position so the substrate 2 is substantiallyparallel to the platform 60A, the set screw 25F can be tightened tosecure the ball joint mechanism 25. As shown, when tightened, the setscrew 25F applies a downward force on the upper socket 25D and thusapplies a frictional force to the top of the ball member 25A via theupper socket 25D. Since the lower socket 25C is fixed to the cap 25E,the force created by the set screw 25F also lifts the lower socket 25Csuch that the lower socket 25C applies a frictional force to the bottomside of the ball member 25A to constrain the ball member 25A within theupper and lower sockets 25C, 25D. Once constrained to the ball member25A, the substrate gripper 20A becomes fixed to the substrate arm 10A.

Typically, once the substrate gripper 20A is positioned and constrainedwith the set screw 25F, the ball joint mechanism 25 need not be adjustedagain during normal use. However, if the substrate gripper 20A becomesmisaligned and therefore the ball joint mechanism 25 requires adjustment(e.g., due to damage, machine repair, poor performance, or otherreasons), the set screw 25F can be loosened, the substrate gripper 20Acan be moved to a closed position to position so that a substrategripped by the substrate gripper 20A is substantially parallel to theplatform 60A, and then set screw 25F can be tightened to secure the balljoint mechanism 25.

In general, actuators 30A and/or 30B can be configured to adjust theposition of substrate arms 10A and/or 10B to vary the extent ofseparation between the surface of platforms 60A and/or 60B and substrate2. Varying this separation provides greater flexibility in embodimentsthat allow for adjusting the fluids assigned to each port, flow rates,fluid viscosities, and evacuation forces from platforms 60A and/or 60B.For example, a 100 micron separation 92 can provide sufficient specimenfixing, staining, and rinsing when fluids applied from platform 60A aredispensed at a flow rate of 70 microliters per second from ports 40A-45Ahaving port diameters ranging from 500 microns to 1,500 microns.Alternatively, with a separation 92 distance between the surface ofplatform 60A and substrate 2 of approximately 200 microns, a higher flowrate for fluids dispensed from ports 40A-45A, such as 115-140microliters per second, can be used for specimen processing.

As disclosed above, machine 1 may contain a series of ports and tubesfor dispersing and removing fluids applied during specimen processing.The following discussion describes various ports, tubes, and othercomponents associated with platform 60A, but similar considerationsapply to platform 60B and its associated components. FIG. 2 shows aclose up view of the apparatus shown in FIG. 1, and shows in detailports 40A-45A on platform 60A and tubes 50A-55A connected to block 80A.Tubes 52A-55A distribute certain fluids including one or more fixatives,stains, and rinse solutions across the platform, into the separation,and onto the substrate.

Referring to FIG. 2, the top side of platform 60A includes six ports40A-45A that are connected to tubes 50A-55A. Fluids are driven by one ormore pumps through the tubes and ports onto substrate 2. One or morefluid reservoirs 210A-213A (such as a first stain reservoir 211A, asecond stain reservoir 212A, a fixative reservoir 210A, and a rinsesolution reservoir 213A), e.g., as shown in FIG. 4, can direct fluidonto platform 60A and substrate 2. The diameters of ports 40A-45A shownin FIGS. 1-3 range from approximately 500 microns to 1,500 microns,although the diameters can also be smaller or larger in certainembodiments. In some embodiments, the diameters of the vacuum ports 40Aand 41A are more than twice the diameters of fluid ports 42A-45A.

Each of ports 40A-45A is typically dedicated to a particular fluid orvacuum source. Alternatively, more than one port may be used for eachfluid or vacuum source, or multiple tubes from various fluid and vacuumsources may connect to a single port located on platform 60A. Forexample, in some embodiments, only one port on platform 60A may be usedfor waste removal, but when using more viscous fluids, the single portmay not provide sufficient suction to evacuate residual fluid from theplatform. Thus, it may be desirable in certain embodiments to providetwo suction ports at different positions on the platform (e.g., onesuction port at each end of the platform) for removing excess stain,fixative, and rinse fluids as shown with ports 40A and 41A in FIG. 2.Further highlighting the variability of fluid-to-port configurations, incertain embodiments, a single port on platform 60A may be dedicated fora particular stain, while in other embodiments multiple ports are usedfor applying stains during specimen processing. Indeed, variouscombinations relating to the number of ports, port locations, and fluidsassigned to each port and fluid tube may be used in differentembodiments of the invention.

Ports 40A-45A can generally be positioned as desired on platform 60A toprovide for fluid delivery to, and fluid removal from, substrate 2.Typically, each of the fluid ports is positioned on platform 60A suchthat the port's aperture is not positioned directly adjacent or beneathspecimen 3 on substrate 2 when the specimen is undergoing processing.With certain combinations of specimens and stains, for example, ifstains are dispensed from a port located directly adjacent or beneath aportion of specimen 3, a larger quantity of stain may be applied tocells in that portion (in the vicinity of the port) than to cells inother portions of the specimen. As a result, cells receiving the largerquantity of stain may appear darker in specimen images, and thisnon-uniform staining of specimen cells can complicate manual andautomated evaluation of the specimen and introduce errors intodiagnostic measurements and analytical outcomes based on the images.Thus, fluid ports that deliver stain to specimen 3 can be spaced acertain distance from the specimen-containing area of a slide to improvestaining results.

In addition, the use of pairs of ports, e.g., multiple pairs of ports,located opposite each other, can also improve staining uniformity. Forexample, in some embodiments, two ports are used to deliver stain tospecimen 3. The two ports can be located on platform 60A at positionsspaced a certain distance (e.g., are offset) from the edges of specimen3, and located opposite each other in a direction parallel to the shortedges of platform 60A. When stain is dispensed from the two spacedports, a relatively uniform quantity of stain is deposited on the cellsin different regions of specimen 3, and improved staining homogeneity isobserved in specimen images.

Similarly, while ports 40A-45A can generally be positioned as desired toremove excess fluids from the surface of substrate 2 using one or morevacuum sources, in some embodiments ports that are used for fluidremoval are spaced at a distance from positions on platform 60A that aredirectly beneath cells within specimen 3 on substrate 2. Positioningwaste removal ports in this manner (i.e., not directly opposing aportion of specimen 3) reduces the chances that when such ports areactuated to evacuate fluids from substrate 2, cells from specimen 3 areinadvertently damaged or drawn into the fluid removal ports. In certainembodiments, due to the difference in lengths of the long and shortsides of platform 60A, the waste removal ports are spaced apart from theedge of the specimen area and arranged opposite each other along adirection parallel to the long edges of platform 60A.

Fixative Phases

Fluid tubes 52A-55A and 52B-55B can be positioned to deliver fixative toplatforms 60A and 60B, separation 92, substrate 2, and specimen 3 duringspecimen processing. Fixatives that can be used include chemicals usedfor protecting biological samples from decay, and such fixatives canimpede biochemical reactions occurring in the specimen and increase themechanical strength and stability of the specimen. Various fixatives canbe used including, but not limited to, methanol, ethanol, isopropanol,acetone, formaldehyde, glutaraldehyde, EDTA, surfactants, metal salts,metal ions, urea, and amino compounds.

Referring to FIG. 4, one or more fluid tubes 52-55A can be connected toa port inside platform 60A and a respective fixative reservoir 210A. Thefluid tubes may also include a connection to a pump 200A and/or a valvecapable of directing fixatives from the reservoir through the tube and aport located on the platform, and onto a substrate and specimen. As anexample, pump 200A can direct fixative from reservoir 210A through tube54A, through block 80A, out from port 44A, onto platform 60A, into theseparation 92 between the platform 60A and substrate 2, and ontosubstrate 2 containing specimen 3. After applying a specific quantity offixative to substrate 2, a vacuum or other suction source 220A and/or221A can evacuate residual fixative from platform 60A, the separation92, and substrate 2 into waste container 230A and/or 231A via one ormore of ports 40A and/or 41A through waste tubes 50A and 51A.

FIG. 9 shows a flow chart 700 that includes a series of steps forapplying fixative to a specimen. In step 702, a pump (e.g., pump 200A)directs fixative (e.g., methanol) from a reservoir (e.g., reservoir210A) into a fixative tube (e.g., tube 54A). In step 704, the fixativeis directed into port 44A attached to block 80A. Then, in step 706, thefixative is directed out of port 44A in platform 60A. In step 708, thefixative is directed out through port 44A and into separation 92 betweensubstrate 2 and platform 60A. Finally, in step 710, specimen 3 onsubstrate 2 is fixed by the fixative solution.

In some embodiments, pump 200A directs methanol through tube 54A andport 44A, onto platform 60A and into the separation 92 at a flow rate of70 microliters per second for a period of four seconds. A vacuum orother suction source 220A and/or 221A then removes residual methanolpresent in separation 92 and/or on the platform 60A and substrate 2using ports 40A and/or 41A and waste tubes 50A and/or 51A (furtherdescribed below). Next, the pump 200A can again direct methanol throughtube 54A and port 44A, and onto platform 60A at a flow rate of 70microliters per second for a period of four seconds, followed by asecond fluid evacuation process. This process of fixing and evacuatingcan be repeated again, using the same or a different fixative, dependingon the type of biological specimen requiring fixation. Further, machine1 is capable of varying the frequency and flow rates for each fixingphase. Other flow rates sufficient to overcome any surface tension inthe fluid located in separation 92 and fix specimen 3 for furtherprocessing and evaluation can also be used. By adjusting the frequencyand/or flow rate of the fixing phases, machine 1 can achieve optimalfixation for various specimens using several different fixatives.Machine instructions for different types of specimens can be hardwiredor preprogrammed in control unit 5 and selected by a system operator asneeded.

In general, a wide variety of fixatives can be applied to specimensduring fixative phases. For example, 85% methanol can be used as thefixative. For some stains, an ethyl alcohol or formaldehyde basedfixative can be used. Additional fixative formulations that can be usedto prepare the specimen are disclosed, for example, in U.S. ProvisionalPatent Application No. 61/505,011, the entire contents of which areincorporated by reference herein.

Staining Phases

Machine 1 also includes tubes and ports configured to apply one or moredyes or stains to a specimen fixed to a substrate in one or morestaining phases. Staining a specimen increases the contrast of thespecimen when it is viewed or imaged under a microscope or other imagingdevice. Romanowsky stains and/or other dyes or stains can be used,including hematoxylin and eosin, fluorescein, thiazin stains usingantibodies, nucleic acid probes, and/or metal salts and ions. Additionalstain formulations that can be used to prepare the specimen aredisclosed, for example, in U.S. Provisional Patent Application No.61/505,011.

FIG. 10 is a flow chart 800 that includes a series of steps for applyingstain to a specimen.

In step 802, a pump (e.g., pump 201A) directs dye or stain from areservoir (e.g., reservoir 211A) into a stain tube (e.g., tube 52A). Instep 804, the stain is directed into a port (e.g., port 42A) attached toblock 80A. Next, in step 806, the stain flows out of port 42A inplatform 60A. In step 808, the stain flows into separation 92 betweensubstrate 2 and platform 60A and thereafter, in step 810, stainsspecimen 3 on substrate 2.

In some embodiments, multiple tubes and ports can be used to apply stainto specimen 3. For example, a second pump (e.g., pump 202A) can directstain (e.g., the same stain or a different stain from that dispensedfrom reservoir 211A) from reservoir 212A through tube 53A and port 43Aand onto platform 60A. In certain embodiments, two or more fluid tubesmay connect to a shared stain reservoir or pump and/or valve used todirect stain through the ports and onto the platform. Referring back toFIG. 2, tube 52A may deliver red stain, such as a fluorescein dye, tothe platform, substrate 3, and specimen 2. Tube 53A may deliver bluestain, such as a thiazin dye. In FIGS. 1-6, the numbers, locations, andsizes of the ports on platform 60A are selected to optimize theapplication of stain to a specimen fixed to the substrate. If otherstains are selected, a different number, locations, and sizes of portsmay be preferable depending on the viscosity of the stain.

Each of ports 40A-45A (and 40B-45B) can include both an input channelfor receiving fluid and an output channel for outputting fluid. In someembodiments, the output channels of the rinse 45A, fixative 44A, andstaining ports 42A-43A are on the upper surface of platform 60A, and theinput channels of vacuum ports 40A and 41A may be on opposite ends ofthe upper surface of platform 60A. The input channels of the rinse 45A,fixative 44A, and staining ports 42A-43A may be situated on the samelateral side of block 80A, and the output channels of the vacuum ports40A and 41A can be positioned on opposite lateral sides of block 80A.

By way of example and with reference to FIGS. 2 and 10, control system 5instructs a pump (e.g., pump 201A) in step 802 to direct a stain (e.g.,a stain comprising fluorescein dye) from a stain reservoir into fluidtube 52A. In step 804, the stain enters port 42A from the fluid tube.Then, in step 806, the stain leaves port 42A at a flow rate of 140microliters per second, for a five second period, and in step 808, thestain is deposited into separation 92 between platform 60A and substrate2 containing specimen 3. In step 810, specimen 3 on substrate 2 isstained. Following staining, a vacuum or other suction source (e.g.,pumps 220 and/or 221) may then evacuate residual stain present inseparation 92, on platform 60A, and on substrate 3 using ports 40A-41Aand waste tubes 50A-51A.

Machine 1 can be programmed to repeat these staining and evacuationphases after a delay (e.g., a delay of between 3 seconds and 10 seconds,such as a five second delay), following the first staining phase. Asecond pump 202A can be instructed by control system 5 to direct thiazindye from a stain reservoir through fluid tube 53A, out port 43A at aflow rate of 140 microliters per second, and onto platform 60A for aperiod of time, e.g., three seconds. A vacuum or other suction source(e.g., pump 220A and/or 221) may then evacuate residual thiazin dyepresent in separation 92 and/or on platform 60A and/or on substrate 2using ports 40A-41A and waste tubes 50A-51A. As with the fixing phases,machine 1 is capable of varying the frequency, delay times, and flowrates for each staining phase. The flow rate may range, e.g., from 70 to140 microliters per second, or may be smaller or greater than the outerlimits of this range (e.g., 10 to 500 microliters per second) providedthe flow rate is sufficient to overcome any surface tension present inthe fluid located in separation 92 and desirably stain the specimen forthe intended evaluation.

Exemplary stains that can be applied to specimens include, but are notlimited to: Wright-Giemsa stain, Giemsa stains, and Romanowsky stains.Other agents such immunocytochemical reagents or other markers ofspecific cell components can also be applied to specimens.

Waste Fluid Removal

As referenced above, a vacuum or other suction source 220 and/or 221 canevacuate residual fluid from substrate 2, separation 92, and platform60A during or between fixing and staining phases. Referring to FIG. 1,one or more waste tubes can be connected to sides 82A and 84A of block80A. Waste or vacuum tubes 50A and 51A are used to withdraw fluid andsmall particulate matter from platform 60A, separation 92, and substrate2 into a waste container or other location separate from machine 1. Withreference to FIG. 2, waste tubes 51A and 51B may be connected toseparate vacuum sources 220 and 221, and waste containers 230 and 231,at the distal ends of the waste tubes. Alternatively, two or more wastetubes can be connected to a single vacuum source, and the same wastecontainer, as shown in FIG. 4. Waste tubes 50A and 50B may extendthrough pinch valves 90A and 90B, respectively.

A vacuum or other source (e.g., vacuum pump 220 and/or 221) for applyingsuction may be connected to one or more of waste tubes 50A, 50B, 51A,and 51B to draw fluid from the platforms 60A and/or 60B, separation 92,and substrate 2 into waste containers 230 and 231. The vacuum forceapplied within the waste tubes may be equivalent to negative one tonegative ten pounds per square inch (“psi”) to provide sufficientsuction for removing fluids when the separation between the substrate 2and the platform is between 100 to 200 microns. In general, as usedherein, “negative” pressure refers to a pressure less than the ambientpressure within machine 1 or the environment surrounding machine 1. Forexample, in some embodiments, the environment surrounding machine 1 hasan ambient air pressure of approximately one atmosphere. “Negative”pressures refer to pressures that are less than this ambient airpressure (e.g., a pressure of negative one psi applied to a fluid is apressure of one psi less than the ambient air pressure exerted on thefluid). Other vacuums ranging from negative 0.1 psi to negative 14 psi(e.g., negative six psi), or greater, can be used provided such vacuumsare sufficient to overcome any surface tension in the fluid present inthe separation and remove all residual fluid in the separation and onthe substrate and specimen. In addition, immediately prior to applyingvacuum to evacuate fluids from the separation, actuator 30A can raisethe proximate edge of substrate 2 a distance of 15-35 microns from thespecimen processing position. This increased separation betweensubstrate 2 and platform 60 can improve evacuation of any residualfluids in separation 92 during a vacuum phase.

In some embodiments, control system 5 is configured to vary thefrequency and vacuum applied for fluid removal during specimenprocessing. FIG. 11A includes a flow chart 900 that features a series ofsteps for removing excess fluid from a substrate. Following a fixingphase, for example, control system 5 can open pinch valves 90A and/or90C in step 902 and apply a vacuum of negative 5 psi in the waste tubes(e.g., waste tubes 50A and 51A) for a five second period. During thisperiod, fixative is removed (step 904) the separation, substrate, andplatform through ports 40A and 41A. The fluid travels through the wastetubes in step 906, and is deposited in into one or more waste containers(e.g., containers 230 and/or 231) in step 908. Once the evacuationperiod expires, control system 5 can instruct one or more of the pinchvalves 90A, 90C to close off the waste tubes 50A and/or 51A in step 910,thereby preventing further evacuation by the vacuum 220-221. Controlsystem 5 may direct machine 1 to repeat this fluid removal step aftereach fixing phase.

FIG. 11B includes a flow chart 1000 that features an alternate series ofsteps for removing excess fluid from a substrate. The method in flowchart 1000 does not use pinch valves to seal waste tubes. Instead, aftera fluid application phase, suction source 220 and/or 221 are initializedin step 1002 and enter an active state in step 1004. The suction sourceapplies a vacuum of negative 3 psi in waste tubes 50A and/or 51A for afour second period to remove fluid from separation 92, substrate 2, andplatform 60A through ports 40A and 41A in step 1006. The evacuated fluidtravels through waste tubes 50A and/or 51A in step 1008, and isdeposited in one or more waste containers 230, 231 in step 1010. Machine1 may repeat this fluid removal step after each fluid application phase.By varying the frequency and pressure applied during fluid removalsteps, machine 1 may achieve optimal fixing, staining, and rinsing ofbiological specimens.

Pinch values 90A, 90B, 90C, and 90D close off waste tubes 50A, 50B, 51A,and 51B, as shown in FIG. 1. The pinch valves 90A-90D may bemechanically, electrically, hydraulically, or pneumatically actuatedthrough actuators contained within or external to the valves. Pinchvalves 90A-90D operate to prohibit fluid flow through waste tubes 50A,50B, 51A, and 51B. For example, when changing or emptying a full wastecontainer 230 from machine 1, it may be desirable to close the pinchvalves (90A-90D) to prevent leakage of residual fluids present in thewaste tubes. Different valve types or other mechanisms such as clamps orstoppers may be used with embodiments of machine 1 to close the wastetubes 50A, 50B, 51A, and 51B.

Rinsing Phases

Rinse solutions can be applied during specimen processing with machine 1in one or more rinse phases. For example, it may be desirable to removeresidual and/or excess fluids from specimen 3 on substrate 2, separation92, and platforms 60A and/or 60B between fixing phases, between stainingphases, and/or between fixing and staining phases. Rinse solutionscompatible with the present systems and methods include distilled water;buffered, aqueous solutions; organic solvents; and mixtures of aqueousand organic solvents, with or without buffering. Additional formulationsfor rinse solutions that can be used to prepare the specimen aredisclosed, for example, in U.S. Provisional Patent Application No.61/505,011.

FIG. 12 includes a flow chart 1100 featuring a series of steps forrinsing a specimen. In step 1102, a pump (e.g., pump 203A) directs rinsesolution (e.g., comprising distilled water) from a reservoir (e.g.,reservoir 213A) into a rinse tube (e.g., rinse tube 55A). In step 1104,the rinse solution enters port 45A connected to block 80A. In step 1106,the rinse solution flows onto platform 60A through the output channel ofport 45A, and in step 1108, the rinse solution enters separation 92between substrate 2 and platform 60A. In step 1110, rinsing of specimen3 is performed. Finally, in step 1112, a vacuum source 220, 221 appliessuction to one or more of waste tubes 50A and 51A to remove rinsesolution from separation 92 and substrate 2; the rinse solution istransported to waste container 230 and/or 231.

In some embodiments, control system 5 may direct pump 203A to apply therinse solution at a flow rate of, e.g., 70 microliters per second for aperiod of, e.g., five seconds. As with fixing phases, control system 5may vary the duration and flow rate of each rinse phase and the numberof rinse phases. In addition, control system 5 may adjust the placementof one or more rinse phases during specimen processing. Control system 5may, for example, direct that a rinse phase occur once, after completionof all fixing phases, and that a second rinse phase occur once, aftercompletion of all staining phases. Alternatively, rinse phases may beinterspersed between two or more fixing phases or between two or morestaining phases.

Agitation Phases

Specimen processing in certain embodiments may include one or moreagitation phases to disperse fixative, stain, and/or rinse fluidsthroughout separation 92, substrate 2 containing specimen 3, andplatforms 60A and/or 60B during the fixing, staining, and/or rinsingphases. FIG. 13 includes flow chart 1200 that features a series of stepsfor agitating a specimen. Actuator 30A and/or 30B, shown in FIG. 3A, canprovide fine movement adjustment for changing the position of substrate2 relative to platform 60A and/or 60B.

Control system 5 can include software and/or hardware for instructingthe actuator 30A and/or 30B to initiate an agitation phase. Actuator 30Aand/or 30B can be configured to move substrate arm 20A and/or 20B up anddown upon an agitation initiation command from the control system. Theagitation phase may repeat for a predetermined number of agitationcycles. The term “agitation cycle,” as used herein, refers to motionfrom a starting position in an upward direction, followed by movement ina downward direction opposite to the upward direction. In someembodiments, one or more agitation cycles return substrate 2 to thestarting position at the conclusion of each cycle, or at least at theconclusion of some cycles. In certain embodiments, substrate 2 does notreturn to the starting position at the conclusion of some or all of theagitation cycles, but each cycle still includes an upward motionfollowed by a downward motion. Actuator 30A and/or 30B typicallycontinues moving substrate 2 in one or more agitation cycles until astop command is sent to the actuator from the control system 5. Anagitation phase may temporarily increase the separation size (separationdistance) between substrate 2 and the surface of platform 60A and/or60B, and then return the substrate to the specimen processing position.In addition, an agitation phase may include a series of movements thatshift substrate 2 between an angular position relative to the surface ofplatform 60A and/or 60B and the specimen processing position. Surfacetension in the fluids dispensed into the separation between the platformand substrate 2 causes a redistribution of fluid molecules on thesubstrate when the substrate moves from the specimen processing positionduring the agitation phase and can advantageously improve fluiddistribution across the specimen.

Other methods can also be used to move substrate 2 relative to theplatforms during agitation phases. For example, in some embodiments, thepositions of one or more of offsets 70A-D and/or 71A-D (e.g., the amountby which the offsets extend above the surfaces of platforms 60A and/or60B) can be rapidly adjusted to agitate specimen 3. In certainembodiments, the positions of platforms 60A and/or 60B can be adjustedto cause agitation of specimen 3. For example, platforms 60A and/or 60Bcan be moved alternately up and down (e.g., corresponding to thedirection of movement of substrate 2 described above) to cause agitationof specimen 3.

In some embodiments, agitation of specimen 3 can be effected by varyingthe extent to which actuator 30A and/or 30B drives substrate 2 towardsoffsets 70A-D and/or 71A-D when the substrate arms are made of amaterial that flexes, as discussed below. Strain gauges can be used tomeasure and adjust the frequency of the agitation applied to substrate 2by detecting the variation in strain in the substrate arms as a functionof time.

Referring to FIG. 13, in a first step 1202, an agitation phase isinitiated. In step 1204, control system 5 instructs actuator 30A tobegin an agitation cycle. In response to this instruction, actuator 30Arotates substrate 2 upward in step 1206, increasing the distance betweensubstrate 2 and platform 60A. Then, in step 1208, actuator 30A rotatessubstrate 2 downward toward platform 60A, reducing the distance betweenthe substrate and platform 60A. In decision step 1210, if the agitationphase is to continue, control returns to step 1204 and the rotation ofsubstrate 2 by actuator 30A occurs again in another agitation cycle. Ifthe agitation phase is to terminate, then control passes from step 1210to step 1212, where substrate 2 is returned to its initial position withagitation complete.

The agitation phase can include one or more agitation cycles appliedthrough actuator 30A and/or 30B. Further, agitation phases can occuronce or multiple times during each of the fixative, stain, and/or rinsephases and in varying frequencies between each of the fixing, staining,and/or rinsing phases. For example, and referring to FIG. 3A, actuator30A and/or 30B may raise the proximate edge of substrate 2 vertically adistance of 35 microns from the specimen processing position andsubsequently return substrate 2 to the specimen processing positionthree times, once after each fixing, staining, and rinse phase. Actuator30A and/or 30B may complete each agitation cycle in two seconds (e.g.,one second to raise the proximate edge of substrate 2 vertically adistance of 35 microns from the specimen processing position and onesecond to return the substrate to the specimen processing position).Machine 1 is capable of carrying out instructions to vary the agitationfrequency and distance for each agitation cycle and/or phase. Forexample, an agitation phase may include actuator 30A and/or 30B raisingthe proximate edge of substrate 2 vertically a distance of 5 micronsfrom the specimen processing position and then returning the substrateto the specimen processing position, 10 to 20 times per second.

Alternative combinations of agitation distances and frequencies can alsobe used. For example, in some embodiments, the agitation distance is 5microns or more (e.g., 15 microns or more, 25 microns or more, 50microns or more, 100 microns or more, 150 microns or more, 200 micronsor more, 250 microns or more, 300 microns or more, 500 microns or more,700 microns or more, 1 mm or more. For example, in certain embodiments,the agitation distance is between 35 microns and 350 microns.

In some embodiments, the agitation cycle frequency is one cycle persecond or more (e.g., two cycles per second or more, three cycles persecond or more, four cycles per second or more, five cycles per secondor more, seven cycles per second or more, ten cycles per second ormore).

Additional agitation techniques can also be used. For example, in someembodiments, substrate gripper 20A and/or 20B may include an actuatorthat rotates the substrate about an axis perpendicular to the rotationalaxis of actuator 30A and/or 30B depicted in FIGS. 1 and 3.

Alternatively, platform 60A and/or 60B may be equipped with an offsetadjuster for raising or lowering the one or more offsets 70A-D and/or71A-D during fixing, staining, and rinsing phases. To implement theoffset adjuster, platform 60A and/or 60B can include offsets that areattached to an internal plate in the platform. The height of the platemay be varied using an internal actuator, thus varying the height of theoffsets. Alternatively, the position of the offsets 70A-D and 71A-Drelative to substrate 2 can be changed by instructing the actuator tomove platform 60A and/or 60B, or block 80A and/or 80B, thereby changingthe separation distance during the agitation phase. Control system 5 canadjust the frequency of fluid cycles, flow rate, offset height,separation distance, and agitation parameters and frequency to processspecimens more efficiently, using significantly less fluid volumesduring the specimen preparation process as compared to conventionalstaining and preparing techniques.

In some embodiments, substrate arms may be made of a material thatflexes such that if a substrate in the specimen processing positionrests against only two offsets extending from the platform, an actuatoror other motive force element may rotate the slide further towards theplatform surface until the slide rests against all four offsets. Varyingthe position of the substrate between these two positions may accomplishsufficient agitation during specimen processing. Substrate arms mayinclude strain gauges to monitor the strain in the substrate arm, andmay be used to inform control system 5 of the position of the substraterelative to the platform offsets. In addition, the control system mayinclude information corresponding to the thickness imperfections of thesubstrate, which the control systems may account for when placing thesubstrate in the specimen processing position or during agitationphases.

Drying Phases

In certain embodiments, the control system 5 can dry the specimen usinga dryer 4 attached to machine 1. FIG. 14 includes a flow chart 1300 thatfeatures a series of steps for drying a specimen. Following the initialstep 1302 in which the completion of the staining and other phases(e.g., one or more rinsing phases) is verified, in step 1304 the dryer 4directs a flow of air across the specimen. The drying process continuesin step 1306, until a signal is received from the control unit to stopthe drying. When the signal is received, the dryer stops the flow of airacross the specimen and the drying phase terminates at step 1308.

In general, machine 1 can be controlled to vary the temperature of theair, the flow rate, the duration of the applied air flow, and thephase(s) during specimen processing for drying the specimen 3. Forexample, after completing a staining phase, dryer 4 can direct a flow ofair at approximately 120° F. at a rate of 10 liters per minute for aperiod of 7 seconds across the specimen. Other air temperatures (e.g.,ambient temperature up to 300° F.), air flow rates (e.g., one liter perminute to 100 liters per minute), and air flow periods (e.g., from a fewseconds to several minutes) can also be used.

Specimen Examination Systems

The automated specimen preparation machines and apparatus disclosedherein, including machine 1, can generally be used with, and/orincorporated into, larger specimen examination systems, such as thosedescribed in U.S. Patent Application Publication No. 2009/0269799, theentire contents of which are incorporated herein by reference. Forexample, FIG. 15 shows a schematic diagram that illustrates one possibleembodiment of a specimen examination system 2000. System 2000 includes aplatform 2100, a light receiving device 2200, a computer 2300, anapplicator 2400, a gas circulation device 2500, a light source 2600, adispenser 2800, a discharge device 2900, a slide labeler 3000, and slidelabel reader 3100. An advancer 2110 may be configured to receive one ormore slides or other substrates 2700. The advancer 2110 may be attachedto a surface, such as the top surface 2101, of the platform. Theadvancer 2110 may take the form of a belt, and the system may use amechanical arm, gravity, magnetism, hydraulics, gears, or otherlocomotion techniques to move substrate-mounted specimens along thesurface 2101 of the platform.

The platform 2100 may also include a feeder 2102 and a collector 2106for respectively feeding and collecting substrates 2700 (e.g., slides)from or to a stack or rack. Feeder 2102 may be equipped with a feederpropulsion mechanism 2103 (such as rubberized wheels) for pushing thespecimens onto advancer 2110. Alternatively, a mechanical arm could beused to grab substrates 2700 and place the substrates on the advancerdirectly. Alternate mechanisms to propel the substrates out of feeder2102 may be used such as magnets or hydraulics. The feeder may include asensor for determining how many slides are present. The sensor couldmeasure the weight of substrates 2700 for example to determine how manysubstrates are present. Collector 2106 can also include a sensor fordetermining how many substrates are present. The sensor can beconfigured to inform the computer 2300 when a preset number of specimenshave been analyzed, and/or can inform the computer of the receipt of aspecimen mounted on a substrate on an ongoing basis.

Light receiving device 2200 can be a microscope (such as brightfieldmicroscope), a video camera, a still camera, or other optical devicethat receives light. Embodiments that include a standard brightfieldmicroscope can also include an automated stage (e.g., a substrate mover2201) and an automated focus. In some embodiments, a microscope can beattached to a motorized stage and a focus motor attachment. Themicroscope can have a motorized nosepiece for allowing differentmagnification lenses to be selected under the control of computer 2300.A filter wheel can be used to enable the computer 2300 to automaticallyselect narrow band color filters in the light path. LED illumination canbe substituted for the filters, and the use of LEDs can reduce the imageacquisition time as compared to the time required for filter wheelrotation. For example, a 1600×1200 pixel FireWire® (IEEE1394 HighPerformance Serial Bus) camera can be used to acquire the narrow bandimages.

In some embodiments, light receiving device 2200 receives lightreflected from substrate 2700 and stores one or more images formed bythe reflected light. Alternatively, or in addition, in some embodiments,fluorescent emission from the specimen on the substrate can be detectedby light receiving device 2200.

In certain embodiments, light receiving device 2200 is configured toobtain transmission images of specimens on substrates. For example,light emission source 2600 can be positioned below the platform and maydirect light so that it passes through platform 2100 and substrate 2700into light receiving device 2200.

Light receiving device 2200 and any of the other components shown inFIG. 15 can be interfaced with the computer 2300 through links(2011-2014), which can provide energy to the component, provideinstructions from computer 2300 to the component, and/or allow thecomponent to send information to computer 2300. Links 2011-2014 can bewired links or wireless links.

Light receiving device 2200 may be capable of X, Y, and Z axial movement(in other embodiments, a motorized stage or substrate mover 2201 mayprovide X, Y, and Z movement). Light receiving device 2200 can includepan, tilt, and/or locomotive actuators to enable computer 2300 toposition light receiving device 2200 in an appropriate position. Lightreceiving device 2200 can include a lens 2210 that focuses incominglight.

Light receiving device 2200 can be selected to capture black and whiteand/or color images. In some embodiments, two or more light receivingdevices can be used to divide the processing time associated withcapturing the images. For example, a low magnification imaging stationcan be followed by a high magnification imaging station. Similarly, insome embodiments, system 2000, platform 2100, computer 2300, and/orlight receiving device 2200 can direct substrate mover 2201 to movesubstrate 2700 to ensure the capture and storage of one or more imagesof all, or most, of the cells on the substrate or on a specific portionof the substrate.

Computer 2300 can be a laptop, a server, a workstation, or any othertype of computing device. The computer can include a processor, adisplay 2320, an interface 2310, and internal memory and/or a diskdrive. Computer 2300 can also include software stored in the memory oron computer readable, tangible media such as an optical drive. Thesoftware may include instructions for causing the computer to operatelight receiving device 2200, applicator 2400, gas circulation device2500, platform 2100, advancer 2110, light source 2600, dispensers 2450and/or 2800, specimen preparation machine 1, or any component within orconnected to one of these components. Similarly, the computer isarranged to receive information from any of these components.

For example, the software may control the rate of dispersal ofsubstrates from the feeder 2102, and feeder 2102 may inform the computerabout the number of substrates present. In addition, computer 2300 canalso be responsible for performing the analysis of the images capturedby light receiving device 2200. Through the analysis process, thecomputer can be arranged and controlled to calculate the number of aspecific type of cell in a particular volume of blood, for example forblood, red cell, white cell, and platelet counts and other measured andderived components of the complete blood count such as: hemoglobincontent, red blood cell morphology, or white blood cell countdifferential could be calculated. The image analysis software cananalyze each individual field and sum the total red and white cellcounts. To calculate the total counts per microliter in a patient bloodsample, the number counted on the slide can be multiplied by thedilution ratio and volume of the sub-sample. Results of the counts,morphologic measurements, and images of red blood cells and white bloodcells from the slide may be shown on the display 2320.

In some embodiments, computer 2300 is configured to display numericaldata, cell population histograms, scatter plots, and direct assessmentsof cellular morphology using images of blood cells displayed on themonitor. The ability to display cellular morphology provides users ofsystem 2000 the ability to quickly establish the presence or absence ofabnormalities in cell morphology that may warrant preparing anadditional slide for manual review by an experienced technician or otherprofessional. The software can also provide the computer withinstructions to display images 2331 received from the light receivingdevice or may cause display 2330 to show the results 2332 (in perhaps achart or graph, for example) of an analysis of the images. Similarly,computer 2300 can be controlled to enumerate the number of cells of aspecific type in a particular blood volume or enumerate the number ofdamaged cells, cancerous cells, or lysed cells in a particular volume ofblood. The software enables the computer to perform the analysisprocess. The computer can use one or more magnifications during theanalysis.

Although shown as one component, computer 2300 can include multiplecomputers; a first computer can be used for controlling the componentsof system 2000, and a second computer can be used for processing theimages from light receiving device 2200. The various computers can belinked together to allow the computers to share information. Computer2300 can also be connected to a network or laboratory information systemto allow the computer to send and receive information to othercomputers.

In certain embodiments, applicator 2400 can include a syringe, a manualor motor driven pipettor, or a motor-controlled pump attached through atube to a pipette tip. Applicator 2400 applies a specimen to substrate2700 in controlled fashion. Exemplary features, attributes, and methodsof using applicator 2400 are disclosed, for example, in U.S. PatentApplication Publication No. US 2009/0269799. The specimen can includeone or more blood components, cells, tissue, or other biologicalcomponents.

Once the specimen has been applied to substrate 2700, the appliedspecimen is processed using machine 1. Machine 1 functions as describedherein to apply one or more stains, fixatives, and/or other solutions tothe specimen on the substrate.

In some embodiments, system 2000 can be configured to achieve minimaloverlapping between cells deposited on substrate 2700 by laying downnon-touching rows of cells from the tip of applicator 2400. Increasingviscosity of the diluted fluid or the type or amount of diluent mayaffect the width of the final settlement positions of specimen flowsfrom the applicator. By selecting a distance between rows to allow forthe typical variation in blood samples, all cells can be counted in allsamples.

Gas movement device 2500, which can be a separate device as shown inFIG. 15, or can be incorporated into machine 1 as discussed previously,can include a fan and/or may include other gas movement devices such asa compressor or a bellows for example. Gas movement device 2500 may beconnected directly to the computer 2300 or may be connected throughanother component such as platform 2100 or applicator 2400. The gasmovement device pushes gas (in some cases atmospheric air) across thesubstrate to control the rate at which substances on the substrate dry.Moving too much air too quickly (i.e., too high of a fan speed) acrossthe substrate can cause cells in the specimen to burst due to rapiddrying, and too little air too slowly (i.e., too low of a fan speed)across the substrate can cause the cells to dry too slowly and appear toshrink.

Computer 2300 can select and control the amount of air that moves acrossthe substrate in a period of time (i.e., the cubic feet or cubiccentimeters of air per second) based upon the distance the gas movementdevice is from the substrate, the type of fluid being analyzed, thewidth of the flows, the temperature of the gas (e.g., air), and theaverage thickness of the flows. Gas movement device 2500 can bepositioned so that the device directs gas so that the gas strikes thesubstrate at an angle of 30°-60° (e.g., 45°) for a period of about 15 to20 seconds. In some embodiments, computer 2300 can control humidity andtemperature settings in the vicinity of the system to allow the dryingprocess to occur without the use of a gas movement device 2500.

Light emission device 2600, and the various components thereof, aredescribed by way of example in U.S. Patent Application Publication No.US 2009/0269799. Various wavelengths of light can be generated by lightemission device 2600 and detected by light receiving device 2200. Forexample, wavelengths such as 415 nm are useful for obtaining ahemoglobin-only image for assessing RBC morphology and hemoglobincontent. Light emitted at 600 nm may be useful to provide high contrastimages for platelets and nuclei. Other wavelengths may be chosen inorder to best discriminate the colors of basophils, monocytes,lymphocytes (all shades of blue), eosinophils (red), and neutrophils(neutral color).

EXAMPLES

The disclosure is further described by the following examples, which arenot intended to limit the scope of the invention recited in the claims.

Example 1

FIG. 16 is a flow chart 1400 showing a series of exemplary steps forprocessing a specimen mounted on a substrate. The steps in flow chart1400 can be used to prepare a biological specimen for examination.Although the description of this process at times refers to specificsteps having specific ranges, and/or discloses steps occurring in aspecific sequence, this description is intended solely as a non-limitingexample. With reference to FIG. 16, machine 1 is connected to a controlsystem 5 for commanding the operation of various machine componentsduring the processing steps. In a specimen initiation step, a biologicalspecimen 3 that includes red blood cells, white blood cells, andplatelets from an aliquot of blood is applied to a substrate 2consisting of a glass microscope slide. This can be performed using adifferent station such as one or more of the stations described inco-pending U.S. Patent Application Publication No. 2008/0102006. In apositioning step 1402, substrate 2 containing specimen 3 is loaded ontosubstrate gripper 20A of substrate arm 10A as shown in FIG. 1. Controlsystem 5 instructs suction source 222 (step 1404) to evacuate air fromthe substrate gripper 20A. Suction applied through suction ports 21 and22 (step 1406) adheres the substrate 2 to the substrate gripper 20Aduring specimen processing. Control system 5 instructs (step 1408) theactuator 30A to rotate the substrate 3 from an open position shown inFIG. 1 to a specimen processing position shown in FIG. 3A. In thespecimen processing position, specimen 3 faces the surface of platform60A while substrate 2 rests against offsets 70A-D shown in FIG. 2. Theoffsets prevent the substrate 2 from making contact with the surface ofplatform 60A. In this example process, the separation 92 between thespecimen-containing surface of substrate 2 and the surface of platform60A is approximately 100 microns.

During the fixation phase (step 1412, see also FIG. 10), a pump appliesfixative to the specimen 3 in step 1414. Pump 200A connected to fluidtube 54A shown in FIG. 2 propels fixative comprising methanol from afixative reservoir 210 through tube 54A, out port 44A, onto platform60A, onto substrate 2 containing specimen 3, and into the separation 92between platform 60A and substrate 2. Pump 200A propels methanol fromport 44A at a flow rate of 70 microliters per second for a two secondperiod T1, thereby directing a total of 140 microliters of methanol, V1,onto substrate 2 containing specimen 3.

Next, in a first agitation step 1416, control system 5 agitates thesubstrate by directing actuator 30A (step 1418) to raise the proximateedge of substrate 2 vertically a distance of 35 microns from thespecimen processing position and returning the substrate to its specimenprocessing position. Machine 1 repeats this agitation step four moretimes. The machine 1 completes the five agitation movements inapproximately ten seconds, T2, as shown in FIG. 17. After agitation, thecontrol system initiates a vacuum or evacuation step 1420. A vacuumforce of negative five psi is applied for one and a half seconds, T3,evacuating any residual methanol (step 1422) present in the separation,on the platform, or on the substrate via ports 40A and 41A, and wastetubes 50A and 51A. The evacuated methanol is collected in a wastecontainer 230 and/or 231.

Following the fixing phase, control system 5 initiates (step 1424) afirst staining phase. In doing so, control system 5 directs the machine1 to stain the specimen (step 1426). Referring to FIG. 2 and theflowchart of FIG. 11, pump 201 connected to fluid tube 52A propelsfluorescein dye from a stain reservoir 211A out port 42A, onto platform60A, onto substrate 2 containing specimen 3, and into the separation 92between the platform 60A and substrate 2. Pump 201 dispenses fluoresceindye through port 42A at a flow rate of 70 microliters per second for atwo second period, T4, thereby directing 140 microliters of dye, V2,onto the substrate.

After applying fluorescein dye to specimen 3, machine 1 performs asecond agitation step 1428 by directing actuator 30A to raise, in step1430, the proximate edge of substrate 2 vertically a distance of 35microns from the specimen processing position and then return thesubstrate to its specimen processing position. Control system 5 causesthe machine 1 to repeat this agitation step two more times and completethe three agitations over a period of approximately six seconds, T5, asshown in FIG. 17.

Next a second vacuum or evacuation phase is initiated in step 1432. Avacuum of negative five psi applied for three seconds, T6, in step 1434to evacuate any residual fluorescein dye present in the separation 92 oron the platform and substrate via ports 40A and/or 41A, and waste tubes50A and 51A. The evacuated fluorescein dye is collected in a wastecontainer 230A and/or 231 A.

After staining the specimen with fluorescein dye, machine 1 initiates asecond staining phase in step 1436 using thiazin dye. Pump 202 connectedto fluid tube 53A propels thiazin dye from a stain reservoir throughport 43A, onto platform 60A, onto substrate 2, and into the separation92 between platform 60A and substrate 2 (step 1438). Machine 1 dispensesthiazin dye through port 43A at a flow rate of 70 microliters per secondfor a two second period, T7, thereby directing a total of 140microliters of thiazin dye, V3, onto the substrate.

After applying stain to specimen 3, machine 1 initiates a thirdagitation phase in step 1440 by directing actuator 30A to raise theproximate edge of substrate 2 (step 1442) a distance of 35 microns fromthe specimen processing position and then return the substratecontaining specimen 3 to its specimen processing position. Machine 1repeats this agitation step three more times. The machine completes thefour agitation movements over a period of approximately eight seconds,T8.

A third vacuum or evacuation step 1444 is then initiated. A vacuum ofnegative five psi is applied for two seconds, T9, to evacuate residualthiazin dye in step 1446 present in the separation or on the platform60A and substrate 2 via ports 40A and/or 41A, and waste tubes 50A and/or51A, after agitation. The evacuated thiazin dye is collected in a wastecontainer 230A and/or 231A.

Machine 1 then performs two rinse-agitation-vacuum phase sequences. Thefirst sequence of phases is initiated at step 1448 when control system 5instructs machine 1 to initiate a first rinse phase. A reservoir 213Acontaining rinse solution of distilled water is connected to a pump 203and fluid tube 55A. Pump 203 directs distilled water through wash tube55A that feeds into port 45A, into the separation 92, and onto platform60A and substrate 2 to rinse specimen 3 in step 1450. Alternatively, insome embodiments, wash fluid is directed through two or more of fluidports 42A to 45A. Pump 203 directs distilled water out of ports 45A at aflow rate of 70 microliters per second for two seconds, T10, therebydirecting a total of 140 microliters, V4, of water onto the substratecontaining the specimen.

Next, control system 5 initiates a fourth agitation phase in step 1452,directing actuator 30A (step 1454) to raise the proximate edge ofsubstrate 2 vertically a distance of 35 microns from the specimenprocessing position and returning the substrate to its specimenprocessing position. Control system 5 may direct the machine 1 to repeatthis agitation phase, and complete the two agitations in approximatelyfour seconds, T11.

Then, a vacuum or evacuation phase is initiated in step 1456. A vacuumof five psi applied for five and a half seconds, T12, in step 1458,evacuates residual distilled water present in the separation 92 or onthe platform 60A and substrate 2 via ports 40A and/or 41A, and wastetubes 50A and/or 51A after agitation.

Thereafter, in step 1460, control system 5 directs machine 1 to beginthe second rinse-agitation-vacuum phase sequence by initiating a secondrinse phase. A second rinse phase (steps 1460, 1462), a fifth agitationphase (steps 1464, 1466), and a fifth vacuum phase (steps 1468, 1470)are performed in the same manner as disclosed above for the firstrinse-agitation-vacuum phase. During the second rinse-agitation-vacuumphase, the amount of wash fluid, V5, and the processing times T13, T14,and T15 are generally the same as in the first rinse-agitation-vacuumphase sequence.

After the specimen has been fixed, stained with fluorescein and thiazinstains, and rinsed, machine 1 initiates a drying phase in step 1472.Dryer 4 directs an air flow of approximately 120° at a 10liter-per-minute flow rate (step 1474) for an eight second period, T16,across the specimen.

Following completion of these steps, substrate 2 is returned to itsoriginal position in step 1476. In this step, actuator 30A rotatessubstrate 2 from the specimen processing position to the open positionas depicted in FIG. 1. Substrate 2 may then be removed by a substratemover, and a new substrate may be loaded for processing a new specimen.

Example 2

The processing steps described above for Example 1 may be adjusted inother embodiments of the invention as follows. In addition, fixative,stains, and rinse solution formulations disclosed in U.S. ProvisionalPatent Application No. 61/505,011 can be used in the following exampleprocessing steps.

During a first fixation phase (step 1412, see also FIG. 10), a pumpapplies a fixative solution to the specimen 3 in step 1414. Pump 200Aconnected to fluid tube 54A shown in FIG. 2 propels a fixative solutioncomprising methanol from a fixative reservoir 210 through tube 54A, outport 44A, onto platform 60A, onto substrate 2, and into the separation92 between platform 60A and substrate 2. Pump 200A propels the fixativesolution from port 44A at a flow rate of 115 microliters per second fora two second period T1, thereby directing a total of 230 microliters ofthe fixative solution, V1, onto substrate 2.

Next, in a first agitation step 1416, control system 5 agitates thesubstrate by directing actuator 30A (step 1418) to raise the proximateedge of substrate 2 vertically a distance of 35 microns from thespecimen processing position and returning the specimen to its specimenprocessing position. Machine 1 repeats this agitation step five moretimes. The machine 1 completes the six agitation movements inapproximately 12 seconds. After agitation, the control system initiatesa vacuum step 1420. A vacuum force of negative six psi is applied forone and a half seconds, T3, evacuating any residual fixative solution(step 1422) present in the separation, on the platform, or on thesubstrate via ports 40A and 41A, and waste tubes 50A and 51A. Theevacuated fixative solution is collected in a waste container 230 and/or231.

Thereafter, in a second fixation phase including a second agitationstep, the foregoing steps of the first fixation phase and firstagitation step are repeated.

Following the fixing phases, control system 5 initiates (step 1424) afirst staining phase. In doing so, control system 5 directs the machine1 to stain the specimen (step 1426). Referring to FIG. 2 and theflowchart of FIG. 11, pump 201 connected to fluid tube 52A propels afirst stain solution comprising eosin Y from a stain reservoir 211A outport 42A, onto platform 60A, onto substrate 2 including specimen 3, andinto the separation 92 between the platform 60A and substrate 2. Pump201 dispenses the first stain solution through port 42A at a flow rateof 115 microliters per second for a two second period, T4, therebydirecting 230 microliters of the first stain solution, V2, onto thesubstrate.

After applying a first stain solution to specimen 3, machine 1 performsa second agitation step 1428 by directing actuator 30A to raise, in step1430, the proximate edge of substrate 2 vertically a distance of 35microns from the specimen processing position and then return thespecimen to its specimen processing position. Control system 5 causesthe machine 1 to repeat this agitation step two more times and completethe three agitations over a period of approximately six seconds, T5, asshown in FIG. 17.

Next a second vacuum phase is initiated in step 1432. A vacuum ofnegative five psi applied for three seconds, T6, in step 1434 toevacuate any residual first stain solution present in the separation 92or on the platform and substrate via ports 40A and/or 41A, and wastetubes 50A and 51A. The evacuated first stain solution is collected in awaste container 230A and/or 231A.

After staining the specimen with the first stain solution includingeosin Y, machine 1 initiates a second staining phase in step 1436 usinga second stain solution including azure B and methylene blue. Pump 202connected to fluid tube 53A propels the second stain solution from astain reservoir through port 43A, onto platform 60A, onto substrate 2,and into the separation 92 between platform 60A and substrate 2 (step1438). Machine 1 dispenses the second stain solution through port 43A ata flow rate of 115 microliters per second for a two second period, T7,thereby directing a total of 230 microliters of the second stainsolution, V3, onto the substrate.

After applying stain to specimen 3, machine 1 initiates a thirdagitation phase in step 1440 by directing actuator 30A to raise theproximate edge of substrate 2 (step 1442) a distance of 35 microns fromthe specimen processing position and then return the specimen 3 to itsspecimen processing position. Machine 1 repeats this agitation step twomore times. The machine completes the three agitation movements over aperiod of approximately six seconds, T8.

A third vacuum step 1444 is then initiated. A vacuum of negative six psiis applied for two seconds, T9, to evacuate residual second stainsolution in step 1446 present in the separation or on the platform 60Aand substrate 2 via ports 40A and/or 41A, and waste tubes 50A and/or51A, after agitation. The evacuated second stain solution is collectedin a waste container 230A and/or 231A.

Machine 1 then performs two rinse-agitation-vacuum phase sequences. Thefirst sequence of phases is initiated at step 1448 when control system 5instructs machine 1 to initiate a first rinse phase. A reservoir 213Acontaining a rinse solution is connected to a pump 203 and fluid tube55A. Pump 203 directs the rinse solution through wash tube 55A thatfeeds into port 45A, into the separation 92, and onto platform 60A andsubstrate 2 to rinse specimen 3 in step 1450. Alternatively, in someembodiments, rinse solution is directed through two or more of fluidports 42A to 45A. Pump 203 directs the rinse solution out of ports 45Aat a flow rate of 115 microliters per second for two seconds, T10,thereby directing a total of 230 microliters, V4, of water onto thesubstrate.

Next, control system 5 initiates a fourth agitation phase in step 1452,directing actuator 30A (step 1454) to raise the proximate edge ofsubstrate 2 vertically a distance of 35 microns from the specimenprocessing position and returning the specimen to its specimenprocessing position. Control system 5 then directs the machine 1 torepeat this agitation phase three more times, and complete the fouragitations in approximately eight seconds, T11.

Then, a vacuum phase is initiated in step 1456. A vacuum of five psiapplied for five and a half seconds, T12, in step 1458, evacuatesresidual rinse solution present in the separation 92 or on the platform60A and substrate 2 via ports 40A and/or 41A, and waste tubes 50A and/or51A after agitation.

Thereafter, in step 1460, control system 5 directs machine 1 to beginthe second rinse-agitation-vacuum phase sequence by initiating a secondrinse phase. A second rinse phase (steps 1460, 1462), a fifth agitationphase comprising six agitations completed in approximately 12 seconds,and a fifth vacuum phase (steps 1468, 1470) are performed in the samemanner as disclosed above for the first rinse-agitation-vacuum phase.During the second rinse-agitation-vacuum phase, the amount of rinsesolution, V5, and the processing times T13, T14, and T15 are generallythe same as in the first rinse-agitation-vacuum phase sequence. Inaddition, immediately prior to the vacuum phase, actuator 30A raises theproximate edge of substrate 2 a distance of 15-35 microns from thespecimen processing position. This increased separation betweensubstrate 2 and platform 60 improves evacuation of any residual fluidsin separation 92 during the final vacuum phase.

After the specimen has been fixed, stained with a first stain solutioncontaining eosin Y and a second staining solution containing azure B andmethylene blue, and rinsed, machine 1 initiates a drying phase in step1472. Dryer 4 directs an air flow of approximately 120° at a 10liter-per-minute flow rate (step 1474) for an eight second period, T16,across the specimen.

Following completion of these steps, substrate 2 is returned to itsoriginal position in step 1476. In this step, actuator 30A rotatessubstrate 2 from the specimen processing position to the open positionas depicted in FIG. 7. Substrate 2 may then be removed by a substratemover, and a new substrate may be loaded for processing a new specimen.

As illustrated in the example specimen processing steps described above,the systems and methods disclosed herein provide for more efficientspecimen processing by consuming fewer reagents as compared toconventional specimen processing methods including automated and manualspecimen preparation techniques. Referring to Example 2, machine 1consumed less than one and a half milliliters of reagents for fixing,staining, and rinsing the specimen during the exemplary processing steps(e.g., 460 microliters of fixative solution+230 microliters of firststain solution+230 microliters of second stain solution+460 microlitersof rinse solution=1380 microliters of reagents). In some embodiments,more or less than 1380 microliters of fluids can be used during specimenprocessing. For example, the amount of fluid used in processing aspecimen can be approximately 1150 microliters (e.g., by eliminating oneof the rinse phases) or less than 1,000 microliters (e.g., by furthereliminating one of the fixative phases).

With respect to FIG. 17, for Example 1, machine 1 consumed less than onemilliliter of reagents for fixing, staining, and rinsing the specimenduring the exemplary processing steps (e.g., 140 microliters of methanolfixative+140 microliters of fluorescein dye+140 microliters of thiazindye+280 microliters of rinse solution=700 microliters of reagents). Insome embodiments, more or less than 700 microliters of fluids can beused during specimen processing. For example, the amount of fluid usedin processing a specimen can be approximately 560 microliters (e.g., byeliminating one of the rinse phases).

In general, the total volume of fluids consumed can be 500 microlitersor more (e.g., 520 microliters or more, 540 microliters or more, 560microliters or more, 580 microliters or more, 600 microliters or more,650 microliters or more, 700 microliters or more, 750 microliters ormore) and/or 2 mL or less (e.g., 1.5 mL or less, 1.4 mL or less, 1.3 mLor less, 1.2 mL or less, 1.1 mL or less, 1.0 mL or less, 900 microlitersor less).

Referring to FIG. 17 and Example 1, the specimen preparation process iscompleted in slightly more than one minute (e.g., 13.5 seconds elapsedduring the fixing phase+11 seconds elapsed during the fluorescein dyephase+12 seconds elapsed during the thiazin dye phase+23 seconds elapsedduring the rinse phases+8 seconds elapsed during the drying phase=67.5seconds total elapsed time). In certain embodiments, specimenpreparation can be completed in more, as in Example 2, or less than 67.5seconds. For example, specimen processing can be completed in 180seconds or less (e.g., 150 seconds or less, 120 seconds or less, 90seconds or less, 80 seconds or less, 70 seconds or less, 60 seconds orless, 50 seconds or less, or 40 seconds or less).

Further, while the foregoing exemplary process describes processing timefor a single specimen, systems and methods for processing multiplesubstrates (e.g., machine 1 in FIG. 1, configured to process twosubstrates, and/or systems configured to process three or moresubstrates) are capable of processing more than 100 specimens per hour(e.g., between 60 specimens and 120 specimens per hour). Use of thesystems and methods disclosed herein in laboratory settings can resultin faster throughput on a per specimen basis, while consumption offluids (e.g., fixative, stain, and rinse fluids) is reduced compared toconventional automated systems and manual specimen preparationtechniques.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description, the foregoing description isintended to illustrate and not limit the scope of the disclosure, whichis defined by the scope of the appended claims. Other aspects,advantages, and modifications are within the scope of the followingclaims.

1-16. (canceled)
 17. A method of preparing a biological specimen on asubstrate for examination, the method comprising: (a) positioning thesubstrate with respect to a surface so that the biological specimenfaces the surface, and so that the substrate and the surface aresubstantially parallel and form a separation of at least about 100microns; (b) sequentially dispensing (i) a first fixative solution, (ii)a first stain solution, (iii) a second stain solution, and (iv) a firstrinse solution into the separation between the substrate and the surfacein an amount sufficient to contact the specimen and the surface; and (c)after dispensing each one of solutions (i), (ii), (iii), and (iv) instep (b), and before dispensing the next one of solutions (i), (ii),(iii), and (iv) in step (b): performing at least a first agitationcycle, wherein the first agitation cycle comprises changing a distancebetween the substrate and surface while the dispensed solution contactsthe specimen for the duration of the first agitation cycle; and removingthe dispensed solution from the separation and from contacting thespecimen.
 18. The method of claim 17, wherein each sequential dispensingstep comprises dispensing one of the solutions in step (b) at a flowrate of at least 70 microliters per second for no more than threeseconds.
 19. The method of claim 17, wherein the first agitation cyclecomprises: increasing the distance between the substrate and the surfaceby at least ten microns; and decreasing the distance between thesubstrate and the surface by at least five microns.
 20. The method ofclaim 17, wherein removing the dispensed solution comprises applying apressure of at least one pound per square inch less than an atmosphericpressure to the separation for at least two seconds.
 21. A method ofpreparing a biological specimen on a substrate for examination, themethod comprising: (a) positioning the substrate with respect to asurface so that the specimen faces the surface, and so that thesubstrate is positioned to form a separation between the surface and atleast a portion of the substrate of at least about 100 microns; (b)performing a fixing phase comprising: (i) dispensing a fixative into theseparation between the substrate and the surface in an amount sufficientto contact the specimen and the surface; (ii) performing at least afirst agitation phase, wherein the first agitation phase compriseschanging a distance between the substrate and surface while the fixativeis contacting the specimen for the duration of the first agitationphase; and (iii) removing the fixative from the separation and thespecimen; (c) performing a first staining phase comprising: (i)dispensing a first stain into the separation between the substrate andthe surface in an amount sufficient to contact the specimen and thesurface; (ii) performing at least a second agitation phase, wherein thesecond agitation phase comprises changing the distance between thesubstrate and surface while the first stain is contacting the specimenfor the duration of the second agitation phase; and (iii) removing thefirst stain from the separation and the specimen; (d) performing asecond staining phase comprising: (i) dispensing a second stain into theseparation between the substrate and the surface in an amount sufficientto contact the specimen and the surface; (ii) performing at least athird agitation phase, wherein the third agitation phase compriseschanging the distance between the substrate and surface while the secondstain is contacting the specimen for the duration of the third agitationphase; and (iii) removing the second stain from the separation and thespecimen; and (e) performing a first rinse phase comprising: (i)dispensing a first rinse into the separation between the substrate andthe surface in an amount sufficient to contact the specimen and thesurface; (ii) performing at least a fourth agitation phase, wherein thefourth agitation phase comprises changing the distance between thesubstrate and surface while the first rinse is contacting the specimenfor the duration of the fourth agitation phase; and (iii) removing thefirst rinse from the separation and the specimen.
 22. The method ofclaim 21, further comprising performing a second rinse phase, the secondrinse phase comprising: (i) dispensing a second rinse into theseparation between the substrate and the surface in an amount sufficientto contact the specimen and the surface; (ii) performing at least afifth agitation phase, wherein the fifth agitation phase compriseschanging the distance between the substrate and surface while the secondrinse is contacting the specimen for the duration of the fifth agitationphase; and (iii) removing the second rinse from the separation and thespecimen.
 23. The method of claim 22, further comprising performing adrying cycle by directing a flow of air across the specimen. 24.(canceled)
 25. The method of claim 21, wherein the combined method stepsare performed in less than 60 seconds.
 26. The method of claim 21,wherein the method consumes less than 650 microliters of fixative, firststain, second stain, and first rinse fluids.
 27. The method of claim 23,wherein the combined method steps are performed in less than 70 seconds.28. The method of claim 23, wherein the method consumes less than 850microliters of fixative, first stain, second stain, first rinse, andsecond rinse fluids.
 29. (canceled)